The preparation of bonded phases to be used as the stationary phase for chromatographic applications has been widely studied. Silanes are the most commonly used surface modifying reagents to prepare bonded phases in liquid chromatography. The chemistry of silanes with various surfaces is well studied. A general discussion of the reaction of silanes with the surface of silicaceous chromatographic support materials is provided in HPLC Columns: Theory, Technology, and Practice, U. D. Neue, Wiley-VCH, Inc., New York (1997). Additional details on the reaction of silanes with porous silicas are disclosed in Characterization and Chemical Modification of the Silica Surface, E. F. Vansant, et al., Elsevier Science B. V. New York (1995). A broad description of the reactions of silanes with a variety of materials is given in Silica Gel and Bonded Phases, Their Production, Properties and Use in LC, R. P. W. Scott, John Wiley & Sons, New York (1993).
The preparation of bonded phases has been described using monofinctional, bifunctional and trifunctional silanes (L. C. Sander et al., (1984) Anal. Chem. 56:504-510). Monofunctional silanes can form only a single covalent bond with silica, thus producing bonded layers having inherently low stability. The bifunctional silanes create bonded layers of somewhat higher stability since they have the capacity to form more chemical bonds. Trifunctional silanes can, in principle, form the greatest number of bonds to the silica surface and hence would be expected to produce the most stable bonded phases. When a trifunctional silylating reagent is employed in place of the monofunctional surface modifying agent, a mixture of ligand surface attachments takes place. These attachments are influenced by the existence of more than a single kind of silanol species on the silica surface, as for example a free silanol (isolated), an associated silanol (vicinal), or a geminal silanol. On the other hand, the trifunctional silane can be attached to the surface by a mono-, bis-, or tris-siloxane bond. The unreacted alkoxy groups, when hydrolyzed to a free silanol, can further react with additional reagent, forming a second layer.
However, it is known that complete removal of all surface silanols is not possible, even when reacting with bi- and trifunctional silanes, because of the randomness of the bonding process and steric hindrance. Most commercially available bonded phases are based on monofunctional silanes because of difficulties in the reproducibility in preparing bonded phases using bi- and trifunctional silanes. Even a small amount of water on the surface of the silica or in the reagents or solvents can substantially increase the amount of bonded phase attached to the surface, resulting in problems in batch to batch reproducibility of the bonded phase. See U. D. Neue, supra, p. 115.
Silica gel has unique properties, which make it highly useful as a chromatographic support, and particularly applicable as a support for high performance liquid chromatography (HPLC). In particular, silica is very popular in HPLC packing because its surface can be modified with a variety of ligands resulting in bonded phases of good mechanical, thermal and chemical stability. Silica gel is the polymeric form of silicic acid, Si(OH)4, in which siloxane bonds are formed between neighboring silicon atoms by eliminating water molecules. Wherever a break in the polymer structure occurs, a silanol group (Si—OH) is present. The surface density of silanol groups on silica gel is about 8 μmole/m2. These silanol groups react with the silylating reagents. Even with the most aggressive silanization reactions no more than 50% of the silanol groups can be converted to silylated derivative because steric hindrance prevents a denser coverage of the surface. Thus, a significant portion of the original silanol groups remain, and these interact with silanophilic analytes, such as basic analytes (generally amines), during chromatographic separations. The presence of unreacted silanol groups also lead to the adsorption of basic analytes on the column, resulting in tailing and asymmetrical peaks or even the irreversible adsorption of the analyte.
Another disadvantage of silica-based bonded phases relates to pH stability. Conventional silica gel based packing materials have limited range of pH stability (2.5-7.5). At low pH, the silicon-carbon bonds break down leading to the erosion of the bonded phase. At high pH, the silica gel itself dissolves, resulting in a loss of bonded phases. In both these instances, there is degradation and irreproducibility in the chromatographic profile. Generally, the pH must be maintained at a prescribed pH, or the column undergoes irreversible damage losing its efficiency and characteristics, such as the ability to produce narrow peaks, desirable retention volumes or resolve components of a mixture. This damage can occur even if the mistaken use outside the narrow pH range defined for the column is only for a short period of time. Typically, bonded phases have a limited lifetime of a few hours, if operated at extremes of pH, to several months if operated under mild conditions. (see R. P. W. Scott, supra, p. 173). Reproducibility in the preparation of bonded phases is important to insure the continued adequacy of the bonded phase for particular separations and separation protocols, which is especially important in forensic analyses or other analytical procedures.
Partial solutions for these problems have been described, such as endcapping to remove residual silanol residues, addition of organic modifiers to the mobile phase, the use of low pH mobile phase to protonate the silanols, introduction of bulkier substituents on the silicon atom of the silane reagent in place of the methyl groups, use of bidentate ligands, formation of silicon-carbon bond in place of the normal siloxane bond between the silica and silane silicon atoms, and the use of mixed trifunctional silanes. Nevertheless, the deleterious effect of surface silanol has not been resolved to the satisfaction of practicing chromatographers.
Another partial solution to the problem of residual silanols is encapsulation of the silica support. Nonpolar linear polymers may be adsorbed onto the silica surface, followed by gamma-ray irradiation to initiate crosslinking. This yields a permanent, nonextractable coating. Such encapsulated silica or alumina supports show high efficiency and resolution for basic silanophilic compounds. Shiseido Company of Japan held encapsulation to be responsible for the superior resolution they report having observed for basic amino analytes on its S/S-C18 reversed-phase packing. However, the preparation of these materials is problematic.
A useful solution to the problem of the residual silanol groups interacting with the analytes is to generate a functionality on the modified silica surface that can react with the silica silanol through electrostatic and/or hydrogen-bonding interaction. Modification of bonded γ-aminopropyl groups by acyl chlorides, active esters, or isocyanates is well documented. A method of the acylation of a pre-formed aminopropylsilylated silica surface to prepare silica-based phase transfer catalysts carrying the acylaminoalkyl chain has been developed (P. Tundo et al., (1979) J. Amer. Chem. Soc. 101:6606-6613). An analogous surface modification procedure has been utilized to prepare an acylaminoalkylsilylated silica stationary phase suitable for chiral liquid chromatography (N. Oi, et al., (1983) J. Chromatogr. 259:487-493). The acylation reactions of aminopropylsilica with a variety of acid chlorides have been studied extensively by A. Nomura, et al., (1987) Anal. Sci. 3:209-212). This study was followed by the work of Buszewski and coworkers with extensive solid state NMR and chromatographic studies on similar acylamino derivatized silicas, termed “peptide bond carrying silicas” by the authors (see B. Buszewski, et al., (1991) J. Chromatogr. 552:415-427). Ascah et al. used a similar chemistry to develop Supelcosil ABZ, which was the first commercial polar-embedded phase (see T. Ascah et al., (1990) J. Chromatogr. 506:357-369). An analogous functionalized silica surface carrying urethane functionalities instead of amide moieties has also been reported (see J. E. O'Gara, et al., (1999) Anal. Chem. 71:2992-2997).
With the incorporation of a polar functional group in the alkyl ligand close to the surface of the silica gel, the phase remains solvated by water at low percentages of organic modifier and even with 100% water. Under these conditions, the alkyl chains maintain their conformational freedom and can interact with polar analytes. The presence of the polar functionality close to the surface acts to shield the effects of unreacted silanol groups. However, because this approach has two individual bonding steps, the phases contain some fraction of unreacted aminopropyl groups in addition to the alkylamide bonded ligands, an alkylester bonded ligands coming from the reaction of acyl chloride and the silanol on silica surface, and residual silanols. The possibility of mixed derivatized and underivatized groups led to potential mixed modes of separation. In addition, due to the fact that this is a side reaction of the desired stationary phase synthesis, the level of residual amino groups is difficult to control. Further, the problem of residual silanol groups, stability of the phases to acid and base, and reproducibility in preparation of the phases, remain unresolved.
These deficiencies in the art have been much improved by the stationary phases and methods of preparation of the invention, as described below.